Association between forage mycotoxins and liver disease in horses

Abstract Background Outbreaks of liver disease in horses are common but the etiology of most remains unknown. Forage mycotoxins have been suspected to be a cause. Objectives To examine the association between outbreaks of liver disease and the presence of mycotoxins in forage stored on the same premises. Animals Premises were identified where ≥4 horses were contemporaneously affected by liver disease, and a control group was formed from premises where ≥4 horses had been examined and found to have no evidence of liver disease. Methods Forage was collected from 29 case and 12 control premises. The forage was analyzed for mycotoxin content using a liquid chromatography/mass spectrometry method, targeting 54 mycotoxins. The presence and distribution of mycotoxins between case and control samples was compared. Results Mycotoxins were found in 23/29 (79%) case samples and 10/12 (83%) control samples (P > .99; relative risk, 0.93; 95% confidence interval [CI], 0.64‐1.75). Median (interquartile range [IQR]) total mycotoxin concentration was similar in case and control samples (85.8 μg/kg [1.6‐268] vs. 315 μg/kg [6.3‐860]; P = .16). Ten mycotoxins were found exclusively in case premises comprising fumonisin B1, 15‐acetyldeoxynivalenol, deoxynivalenol, zearalenone, aflatoxins B1 and G1, methylergonovine, nivalenol, verruculogen, and wortmannin. The median (IQR) concentration of fumonisin B1 was significantly higher in case versus control samples (0 μg/kg [0‐81.7] vs. 0 μg/kg [0‐0]; P = .04). Conclusions and Clinical Importance Several mycotoxins with known hepatotoxic potential were found, alone or in combination, exclusively at case premises, consistent with the hypothesis that forage‐associated mycotoxicosis may be a cause of outbreaks of liver disease in horses in the United Kingdom.


| INTRODUCTION
Liver disease is commonly encountered in equine practice both as clinical and subclinical disease. 1 Outbreaks of hepatic disease are common and once often were suspected to be caused by pyrrolizidine alkaloid (PA) toxicosis, 2 although more recent evidence suggests that PA toxicity is far less common than generally suspected. 3 Some other plants also have been incriminated in hepatopathy cases although incidents appear to be rare. 4 Alternative explanations for outbreaks of hepatopathy might include infectious pathogens, and evidence recently has emerged of viral hepatitides in horses. 5,6 However, in the majority of liver disease outbreaks in horses, specific etiologic diagnoses remain elusive. Many fungi produce metabolites that possess antibacterial, antiviral, anthelmintic, antifungal, herbicidal, and insecticidal properties, which may provide a competitive advantage. 7,8 Some fungal products are also toxic to mammalian species, and >500 such mycotoxins have been identified, primarily from fungi of the genera Aspergillus, Penicillium, Alternaria, and Fusarium. 9 Mycotoxins are a major human health concern with approximately 25% of global crop production being contaminated. 10 Monogastric species including horses are considered more susceptible to mycotoxins than ruminants 11 with the main groups endangering animal health comprising alflatoxins, ochratoxins, trichothecenes, fumonisins, and zearalenone. 10,12,13 As the site for toxin biodegradation, the liver appears especially susceptible to xenobiotics, and several mycotoxins are known to be associated with hepatopathy. 11 Forages comprise the bulk of a typical diet for horses and, anecdotally, outbreaks of hepatopathy are sometimes resolved after a change of forage. Several studies have shown mycotoxins to be common in hay in Europe and the United States (USA), [14][15][16][17] although associations with disease have not been investigated.
We aimed to look for evidence of mycotoxicosis as a possible cause of hepatic disease in horses by comparing the prevalence of different mycotoxins in hay samples from premises known to have an outbreak of hepatopathy in resident horses versus yards with no such evidence.

| METHODS
Our case control clinical study identified premises of horses within a single equine veterinary practice where client-owned resident horses were known to have ("case premises") or not have ("control premises") suffered an outbreak of liver disease between December 2012 and December 2017. As soon as horses were identified for inclusion in the study, hay samples from case and control premises were collected and submitted for mycotoxin analysis using a liquid No. L20-392, Perry Johnson Laboratory Accreditation, Inc]) following methods described previously. 18 Sampling was performed by selecting a handful of hay from the center of 6 to 10 stored bales randomly selected from different areas of the hay store and pooling all of the collected samples into a single plastic bag for analysis by the testing laboratory. The sample was homogenized at the testing laboratory before analysis.
A case premises was defined by at least 4 resident horses being confirmed to have liver disease contemporaneously on the basis of serum biochemistry or liver biopsy with no etiology identified The actual serum biochemical analytes measured varied among cases, but at least included gamma glutamyltransferase (GGT). Typically, an index case was identified after a veterinary examination, and serum biochemical testing requested by the horse's owner because of a clinical concern. In such index cases, testing included GGT, aspartate aminotransferase (AST) and glutamate dehydrogenase (GLDH) activities, often along with several other hepatic and nonhepatic biomarkers such as hematology, serum protein concentration, acute phase protein concentrations, alkaline phosphatase activity, bile acid concentrations, and serum creatinine and urea concentrations.
After diagnosis of the index case by the attending veterinary surgeon, owners of horses also present on the same premises were advised to have serum biochemical investigations performed on their horses to determine whether or not they were part of a wider subclinical hepatopathy outbreak, as is standard procedure for this veterinary practice. Such secondary screening included at least measurement of serum GGT activity and, in some cases, also GLDH and AST activity. Case premises were included where such testing identified serum biochemical evidence of liver disease in at least 4 resident horses and at least 70% of all tested horses were determined to be affected by liver disease (ie, affected/total:

| RESULTS
In total, 29 case premises and 12 control premises were identified and hay samples tested. In case premises, 23/29 (79%) samples were positive for mycotoxins compared to 10

| DISCUSSION
We found that mycotoxins were present in >80% of hay samples fed to horses in the United Kingdom, although neither the overall prevalence of mycotoxins nor total mycotoxin concentration differed between hay fed to horses with liver disease versus those from  16 and the majority of mycotoxins found in grass and hay are known to be produced mainly or exclusively by Fusarium species, including zearalenone, trichothecenes, and fumonisins. 10 Zearalenone and the type A and type B trichothecenes, T2 and deoxynivalenol, were found to be very common in grass in the Czech Republic, especially in July, with lower concentrations in June and December. 19,20 The mass of these mycotoxins was found to increase further during storage of the grass as silage. 19 Similarly, examination of rye grasses in Germany found zearalenone in 67% of samples, and the type A trichothecenes T2 and diacetoxyscirpenol in 25% and 22% of samples, respectively. 16 A more recent study from Germany also found zearalenone to be the most common mycotoxin in hay (43% of samples), with the type B trichothecenes nivalenol, deoxynivalenol and 3-acetyldeoxynivalenol also found in lesser quantities. 15 In Ireland, examination of 149 hay samples, grown locally and imported from Canada, found only zearalenone as a mycotoxin contaminant. 14 It was found in 21% of Irish hay samples but in only 8% of Irish haylage or imported Canadian hay. Thus, zearalenone appears to be the most common mycotoxin in European hays, with trichothecenes also often present. In contrast, a study of hays and silages in Minnesota, Wisconsin, and Illinois in the United States failed to detect any zearalenone although Alternaria alternata TA toxin, cyclopiazonic acid, deoxynivalenol, fumonisin B1 and roquefortine all were very commonly found. 17 The most common mycotoxins found in hay in our study were fusaric acid, ochratoxin A, fumonisin B1, penicillic acid, and neosolaniol ( Adverse health consequences of mycotoxins can be complex and unpredictable and depend on mycotoxin concentration, chronicity, animal species, bioavailability and co-exposure to other mycotoxins. 10,21,22 In addition to the association of fumonisin B1 with liver disease in our study, it is possible that other mycotoxins also might have pathogenetic relevance. Evidence of interactions among several hepatotoxic mycotoxins is reported including aflatoxin B1, deoxynivalenol, nivalenol, T2, fumonisin B1, zearalenone, and moniliformin. 23,24 Interestingly, although less commonly found than fumonisin B1 in our study, aflatoxin B1, 15-acetyldeoxynivalenol, deoxynivalenol, nivalenol, and zearalenone also were found exclusively in hay samples from case premises (Table 1).
Fumonisin B1 acts as a competitive inhibitor of ceramide synthase and thus inhibits the sphingolipid biosynthetic pathway. 25 Horses are considered to be more sensitive to fumonisin than other species and neuroand hepato-toxicity are the main consequences. 26,27 Fumonisin B1 is best known in equine medicine as the cause of leukoencephalomalacia in association with ingestion of moldy corn. 28 However, an outbreak of equine leukoencephalomalacia in horses also has been reported after consumption of fumonisin-contaminated forage. 29 Fumonisin B1 also is known to cause hepatotoxicity in many species including rats, 30 mice, 31 rabbits, 32 pigs, 33 calves, 34 chickens, 35 and horses. [27][28][29]36 Although acute hepatotoxicity with centrolobular necrosis, cytoplasmic vacuolation, biliary hyperplasia and moderate to severe portal fibrosis is recognized in association with ingestion of large amounts of fumonisin B1 in horses, 27 Most cases of leukoencephalomalacia in horses have been associated with feeds containing >10 ppm fumonisin B1, and it is recommended that feeds for horses contain no more than 1 to 5 ppm fumonisin B1. 27 In this respect, only 1 of the cases in our study had a concerning value Various approaches have been used to attempt to decrease mycotoxin presence in feeds using processing or additives, 9,38 although only some would be feasible for forage treatment. Heat may destroy mycotoxins such as fumonisin B1, alflatoxin B1, deoxynivalenol, nivalenol, and zearalenone although often temperatures >150 C are required for a consistent effect, which is probably beyond the range of most hay steamers. [39][40][41] Some mycotoxins such as fumonisin B1, deoxynivalenol, and nivalenol are highly water soluble and may be removed effectively by washing or soaking whereas others such as alflatoxins, zearalenone, and type A trichothecenes are more hydrophobic. 42 Many potential adsorbents are available with variable efficacy for removal of mycotoxins.
Silicates such as kaolin, bentonite, montmorillonite and zeolite are the most widely used although others exist, and charcoal, yeast (including Saccharomyces cerevisiae), and bacteria (including Lactobacilli) also have been studied. 9 Another approach to mitigating the effect of mycotoxins includes nutritional supplements to modulate detoxification of mycotoxins or to counteract their toxic effects. One study found that glucose reactants of fumonisin were far less toxic in pigs. 33 Various vitamins and other micronutrients such as vitamin E, selenium, methionine, glutamic acid, arginine, aspartate, and lysine have been investigated in this respect. 9 Different grass species appear to have different susceptibilities to fungal colonization and mycotoxin production, offering a means of control when mycotoxicosis occurs. 19,20,43,44 Fertilization of pasture also might decrease mycotoxin contamination in some cases. 44 The major limitation of our study was the potential for sampling analysis. Nevertheless, the presence of specific mycotoxins found in our study at least establishes that the criteria for toxinogenesis were fulfilled in specific samples. The study was further limited by availability of premises for inclusion, especially control premises. Thus, statistical power was suboptimal and the descriptive findings simply provide a basis for further study of certain mycotoxins. In particular, mycotoxins that were found more commonly in control premises (eg, fusaric acid, ochratoxin, penicillic acid) would appear unlikely to have relevance to hepatopathy outbreaks and focus on other mycotoxins such as fumonisin B1 would appear more logical (Table 1).
Our study was based on the hypothesis that forage-associated mycotoxins might be a cause of liver disease outbreaks in horses in the United Kingdom. Although the study did not establish causal associations between mycotoxins and hepatopathy, interesting associations between liver disease and mycotoxins with known hepatotoxic potential were found, providing a basis for further investigation of any putative links.

ACKNOWLEDGMENT
Funding provided by Alltech. The author thanks Helen Warren of Alltech Inc. for providing the mycotoxin analyses in this study.

CONFLICT OF INTEREST DECLARATION
Author declares no conflict of interest.

OFF-LABEL ANTIMICROBIAL DECLARATION
Author declares no off-label use of antimicrobials.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION
Author declares no IACUC or other approval was needed.

HUMAN ETHICS APPROVAL DECLARATION
Author declares human ethics approval was not needed for this study.